Biology subjects

Bromme, D.

Publications and source records attributed to Bromme, D..

4 recordsLinked to original sources

bioRxiv2025

Structural Divergence without Functional Impact: Comparative Characterization of SARS-CoV-2 3CL-Mpro Variants Using Cleavage Site Substrates

The SARS-CoV-2 main protease (3CLpro) is essential for viral replication and a leading antiviral target. Circulating variants accumulate substitutions on this enzyme, distant from the catalytic site. Surprisingly, mutant enzymes retain full proteolytic activity, though preserved overall activity does not exclude subtler effects on substrate recognition or selectivity, arising from distal structural perturbations. In this study, we compared the steady-state kinetics of wild-type (Wuhan) 3CLpro with enzymes from the Beta (K90R), Lambda (G15S), and Omicron (P132H) variants, using two peptide substrates representing distinct viral polyprotein cleavage sites. All four proteases displayed comparable catalytic efficiencies, similar pH-rate profiles, suggesting conservation of the catalytic mechanism despite sequence variation. The crystal structure of Omicron 3CLpro bound to an Nsp8-Nsp9 peptide revealed a conserved fold and active-site geometry, with the P132H side chain adopting a substrate-dependent conformation that rebuilt its local contacts, indicating towards how a distal substitution is accommodated without perturbing catalysis. Thermal stability measurements identified the sole distinguishing effect of P132H, with Omicron showing altered stability at elevated temperature. A screen of 31 tanshinones against 3CLpro identified T06 with Ki values of 5 uM, as 3CL pro inhibitor. Thus, 3CLpro may tolerate distal substitutions through local structural adaptation, supporting its durability as an antiviral target.

biochemistry↗

bioRxiv2025

Cathepsin K as a Key SARS-CoV-2 Cell Entry Protease and Dual-Inhibition Target

SARS-CoV-2 relies on host proteases to prime its spike protein for cell entry through either the endosomal or plasma membrane pathway. Although cysteine cathepsins are known to mediate the endosomal route, the identity of the dominant enzyme has remained unclear. Here, we identify human Cathepsin K (hCatK), a lysosomal cysteine protease, as a previously unrecognized yet functionally important mediator of spike activation. While human Cathepsin L (hCatL) has long been regarded as the principal endosomal protease for spike processing, inhibition of hCatK with the selective inhibitor Odanacatib suppressed viral infection in endothelial cells as effectively as the broad-spectrum cysteine protease inhibitor E-64d, implicating hCatK as a key driver of spike processing during the endosomal viral entry. Comprehensive enzymatic profiling demonstrated that hCatK exhibits 24- to 63-fold higher catalytic efficiency toward the Furin-cleavage site (FCS) sequence than hCatL and displays a distinct substrate-recognition pattern at the Omicron FCS relative to the Wuhan variant. We further demonstrate that hCatK is an off-target of Nirmatrelvir, a clinically approved 3CL-Mpro inhibitor, with a sub-micromolar potency (IC50 = 0.6 {+/-} 0.1 {micro}M). A 1.9 [A] crystal structure of the hCatK-Nirmatrelvir complex delineates the molecular basis of inhibitor binding and supports the rational design of dual-acting antivirals. Collectively, these findings redefine the landscape of host proteases involved in SARS-CoV-2 spike activation and establish hCatK as a previously overlooked but strategic target for antiviral intervention.

biochemistry↗

bioRxiv2025

Beyond Degradation: How Reverse Proteolysis Creates Disease-Relevant Antigens

Proteases are conventionally regarded as degradative enzymes, yet their catalytic machinery also permits peptide bond formation through reverse proteolysis, a process that remains poorly characterized. Here, we show that lysosomal cysteine cathepsins catalyze iterative cycles of hydrolysis and ligation to generate multi-generational fusion peptides, including hybrids derived from host-viral protein substrates. Quantitative analysis demonstrates that peptide ligation can account for up to 4.5% of proteolytic turnover. This activity is strongly influenced by pH, substrate sequence, and post-translational modification, with citrullination and neutral pH favoring fusion peptide formation and the accumulation of more stable higher-order products. Using full-length protein substrates, we provide direct evidence that cathepsins can generate a hybrid insulin peptide previously identified as a Type 1 Diabetes (TID) autoantigen in patients. Moreover, several identified fusion peptides show effective binding to TID-associated HLA class II molecules. To examine whether ligation products can be captured under cellular conditions, we developed a click-based targeted transpeptide retrieval and purification strategy (CT-TRAP), which enabled detection of probe-derived cis/transpeptides in cell-based systems under controlled conditions. These findings establish reverse proteolysis by cysteine cathepsins as a quantifiable enzymatic pathway for generating non-genomically templated peptides, revealing an unrecognized dimension of lysosomal protease activity and peptide diversification.

biochemistry↗

bioRxiv2025

Cathepsin K inhibitors promote osteoclast-osteoblast communication and engagement of osteogenesis

Cathepsin K inhibitors are well known for their inhibitory activity against bone resorption but, in contrast with other bone resorption antagonists, were also reported to preserve bone formation in clinical trials. Here we show cathepsin K inhibitors favor the crosstalk between osteoclasts and osteoblasts and help engaging the osteogenic process required for proper bone remodeling. Therefore, we used a novel approach, co-culturing human osteoclasts and osteoblast lineage cells on bone slices and monitored through time-lapse their response to an active site (odanacatib) or an ectosteric (T06) cathepsin K inhibitor. Both inhibitors prevent the shift from pit to trench resorption mode and thus lead to a marked increase in pit-eroded surface lined with undigested collagen. Importantly, pit-eroded surfaces prove to receive significantly more and longer visits of osteoblast lineage cells. Furthermore, resorption achieved under CatK inhibition promotes osteoblast differentiation as shown by upregulation of alkaline phosphatase and type 1 collagen, and down regulation of RANKL. We propose a model where high cathepsin K activity levels lead to both aggressive bone resorption and compromised bone formation, and where low cathepsin K levels result in both slower resorption and faster initiation of formation. This model fits the current knowledge on the effect of collagen/collagenolysis on osteoclast activity and osteoblast chemotaxis. The combined effects of cathepsin K on resorption and formation render cathepsin K inhibitors unique tools to prevent bone loss. They stress the clinical interest of developing ectosteric inhibitors that may limit the side effects of active site inhibitors. LAY SUMMARYSmall bone packages are continuously degraded by osteoclast cells and reconstructed by osteoblast cells. Too much degradation or too little reconstruction leads to bone loss and is currently treated with inhibitors of degradation or stimulators of reconstruction. There is usually little attention for the mechanism maintaining the balance between degradation and reconstruction. This mechanism involves proper communication between osteoclasts and osteoblasts. Here we show that cathepsin K inhibitors developed to inhibit degradation, also favor osteoclast-osteoblast communication, thereby allowing a faster preparation of degraded bone surfaces for new bone deposition. This highlights the unique clinical potential of cathepsin K inhibitors.

cell biology↗